Enhanced Protein Homeostasis Mechanisms in Naked Mole Rats Cells Harrison Pride 1, Alex Coles 2, Jillian Mochnick 3, Yiqiang Zhang 4,5, Rochelle Buffenstein 4,5, Peter J Hornsby 4, 5, Steve Austad 5.6, Arlan Richardson 5,6, and Viviana I. Pérez 3,7 Departments of Microbniology 1 and Biochemistry and Biophysiscs 3, Oregon State University; Department of Chemistry and Biochemistry 2, University of Michigan, Flint, Flint, MI 48502; Departments of Physiology 4, Cell and Structural Biology 5, Barshop Institute for Longevity and Aging Studies 6, The University of Texas Health Science Center, San Antonio, Texas 78249; and Linus Pauling Institute 7, Oregon State University, Corvallis, Oregon Enhanced Protein Homeostasis Mechanisms in Naked Mole Rats Cells Harrison Pride 1, Alex Coles 2, Jillian Mochnick 3, Yiqiang Zhang 4,5, Rochelle Buffenstein 4,5, Peter J Hornsby 4, 5, Steve Austad 5.6, Arlan Richardson 5,6, and Viviana I. Pérez 3,7 Departments of Microbniology 1 and Biochemistry and Biophysiscs 3, Oregon State University; Department of Chemistry and Biochemistry 2, University of Michigan, Flint, Flint, MI 48502; Departments of Physiology 4, Cell and Structural Biology 5, Barshop Institute for Longevity and Aging Studies 6, The University of Texas Health Science Center, San Antonio, Texas 78249; and Linus Pauling Institute 7, Oregon State University, Corvallis, Oregon Introduction Methods Results Conclusions Acknowledgements Abstract Phylogenic studies suggest that cells from long-lived species are more resistant to a variety of stressors than short-lived species. However, there is little information on the cellular mechanisms that give rise to increased resistance to stress. Our previous studies have shown that liver proteins from a long-lived species have lower levels of protein ubiquitination which is associated with increased proteasome activity, suggesting that mechanisms of protein quality control could play a critical role in assuring longevity of long- lived species. In this study, we evaluated whether autophagy and heat shock chaperones proteins (HSPs) are associated with longevity in rodents using skin fibroblasts isolated from mice and naked mole rats (NMR); two species that are similar in body size but differ almost 10 fold in longevity. Our results indicate that macroautophagy induced by serum- starvation is significantly enhanced in NMR compared to mouse which correlates with an inhibition of the mTOR pathway and increased LC3 conversion. We also found that several HSPs (e.g., Hsp90, Hsp70, Hsp40, Hsp 27) were significantly higher at both basal and after heat shock conditions. These observations suggest that NMR, a long-lived species, has increased mechanisms to ensure protein quality (autophagy and HSPs) and support the idea that protein homeostasis could play an important role in promoting longevity. Financial support was provided by Ellison Medical foundation (to both V.P and RB), American Federation for Aging Research and The Glenn Foundation (RB). Naked mole rats (MRs) are unique rodents that hold the record for the longest lifespan, over 28 years. It is believed that the reason for this long lifespan is how their cells handle stress. Our previous studies suggest that mechanisms that are important for protein quality control (protein homeostasis) could play a critical role in longevity of NMR. In this study, we evaluated autophagy and heat shock chaperones proteins (HSPs) and to determine how they may contribute to enhanced longevity, we used skin fibroblast from two clades, i.e., short- and long-lived rodents [mice and naked mole rats (MRs)], and short- and long-lived bats. Cells: Rodents: Primary skin fibroblast from mice [Mus musculus, 35gr and 4 years (y)], and NMRs [Heterocephalus glaber, 30 gr and 30 y], were cultured at 35 0 C and 5% CO2. Bats: evening bat [Nycticeius humeralis,11gr and 6y] shortest-lived bat known v.s little brown bat [Myotis lucifugus,8gr and 34y] were cultured at 37 0 C and 5% CO2. Autophagy measurement: Autophagy was measured by monitoring the degradation of radioactively-labeled long-lived proteins as described by Massey et al. (2010). Macroautophagy was measured in presence of 3-methyladenine (3-MA), and an estimate of the chaperone mediated autophagy (CMA) was obtained by measuring the difference in the rate of protein degradation between that observed in the presence of 3-MA and the rate in the presence of leupeptin plus ammonium chloride. Non-lysosomal protein degradation was measured as the residual value of protein degradation obtained in presence of all three inhibitors under serum or serum-free conditions. Furthermore, we measured of the LC3 ratio (LC3II/LC3I) and p62 level were measured. Heat shock protein: Cell cultures were exposed to a 1 hour heat shock (41 0 C), after which samples were placed at normal temperature (35 or 37 0 C) and collected at 0, 2, 4, 6, 24 hrs thereafter. Cells were lysed, and cellular levels of Hsp 90, Hsp 70, Hsp 40, and Hsp 27, were determined by western blots analysis SerumSerum Free P62 Levels 41% 50% Hour2 Hour4 Hour6 Hour24 Hour HSP Actin (Hr) Evening BatLittle Brown Bat Hour2 Hour4 Hour6 Hour24 Hour HSP Hour2 Hour4 Hour6 Hour24 Hour Hour2 Hour4 Hour6 Hour24 Hour HSP 40 HSP 27 V.S Normalized pS6/S6 (% of Serum) SerumSerum Free SerumSerum Free p62 Levels SerumSerum Free EB LBB Normalized LC3II/LC3I (% of serum) # * LC3II LC3I EBLBB C SFC S6K pS6K EBLBB C SFC p62 actin EBLBB C SFC V.S A B C DE Figure 2 Figure 1 Figure 3 Figure 4 - Macroautophagy induced by serum-starvation is significantly enhanced in fibroblasts from the NMRs compared to mice, and this increase can be correlated with an inhibition of the mTOR pathway as well as an increased conversion of LC3I to LC3II. Similar findings were observed in the long-lived bat (Little brown bat, LBB), where macroautophagy induced by serum-starvation is significantly higher that the short-lived bat (Evening bat, EB). - The heat shock chaperone levels (basal and induced by temperature (41°C) were significantly higher in NMRs compared to mice. This was not the case when we studied heat shock chaperone in bats, where the long-lived bat (LBB) had similar or even lower levels of chaperones when we compared to the short-lived bat (EB). Collectively these data provide further evidences that naked mole-rats have far better protein homeostasis mechanisms than mice and bats. Furthermore our data also suggest that enhanced autophagy may be a longevity determinant since is the common mechanism among these two long-lived species, NMRs and LBB. Our current studies in a third clade, the marsupials, will give allow us to test this hypothesis, where we will expect that long-lived marsupial will have enhanced autophagy then the short-lived one. Figure 1: Macroautophagy induced by serum-starvation is significantly enhanced in fibroblasts from the NMRs compared to mice. Protein degradation in skin fibroblast from NMRS and mice was determined by a pulse-chase technique using [ 3 H]-valine (2μCi/ml) at 24 hours. Total rates of protein degradation in fibroblast from NMRS (open circles) and mice (solid quares) in medium supplemented with 10% FBS (A) or in serum-free medium (B). Protein degradation measured by Macroautophagy (C), Chaperone-mediated autophagy (CMA) (D), and Non-lysosomal protein degradation (E). Also, Macroautophagy was determined also by the ratio of LC3II to LC3I conversion (LC3II/LC3I), (F), mTOR signaling through the ratio of P-S6/S6 (G), and p62 (H), using western blot analysis of total cellular homogenates from NMRS solid bars) and mouse (open bars) under serum and serum-free conditions. The data are the mean ± SEM of triplicate measurements obtained from 3 different animals and were analyzed by the non-parametric test of ANOVA. The asterisk (*) and pound (#) denote a statistically significant difference between NMRS and mouse at the p<0.05 and p<0.01 Figure 3: Heat Shock proteins levels are higher in NMRs than mice. Heat Shock proteins (Hsp) 90, Hsp 70, Hsp40, Hsp 27, were measured by western blotting in whole cell extract. Significant differences in all heat shock proteins were observed in NMRs compared to mice. A big difference was specially observed for those small molecular weight Hsp (e.g., Hsp 27 and Hsp40). This data confirm that NMRs have a better machinery for proteome homeostasis. The data are the mean ± SEM from 3 different animals and were analyzed by the non-parametric test of ANOVA. The asterisk denote a statistically significant difference p<0.05. F G H F G H A B CD E Figure 2: Macroautophagy induced by serum-starvation is significantly enhanced in fibroblasts from the LBB compared to EB. Protein degradation in skin fibroblast from NMRS and mice () was determined by a pulse-chase technique using [ 3 H]-valine (2μCi/ml) at 24 hours. Total rates of protein degradation in fibroblast from LBB (open circles) and EB (solid squares) in medium supplemented with 10% FBS (A) or in serum-free medium (B). Protein degradation measured by Macroautophagy (C), Chaperone-mediated autophagy (CMA) (D), and Non-lysosomal protein degradation (E). Also, Macroautophagy was determined also by the ratio of LC3II to LC3I conversion (LC3II/LC3I), (F), mTOR signaling through the ratio of P-S6/S6 (G), and p62 (H), using western blot analysis of total cellular homogenates from LBB solid bars) and EB (open bars) under serum and serum-free conditions. The data are the mean ± SEM of triplicate measurements obtained from 3 different animals and were analyzed by the non-parametric test of ANOVA. The asterisk (*) and pound (#) denote a statistically significant difference between NMRS and mouse at the p<0.05 and p<0.01 * * * * # MouseNMR Figure 4: Heat Shock proteins levels are similar or lower in LBB (long-lived bat) than EB (short-lived bat). Heat Shock proteins (Hsp) 90, Hsp 70, Hsp40, Hsp 27, were measured by western blotting in whole cell extract. No significant differences or lower levels of Hsps were observed in LBB compared to EB. Our data so far will suggest that heat shock proteins are not elevated in the long-lived bat compared to the short-lived one. The data are the mean ± SEM from 3 different animals and were analyzed by the non-parametric test of ANOVA. The asterisk denote a statistically significant difference p<0.05. MouseNMR Actin (Hr) (Hr)